Ultrasound and acoustophoresis for collection and processing of oleaginous microorganisms

ABSTRACT

Microorganisms such as microalgae are collected and separated from a host medium such as water. Cellular walls and membranes of the microorganisms are then ruptured to release their lipids using a lipid extraction unit. Thereafter, the lipids from the host medium are collected and separated using a lipid collection and separation unit. Related apparatus, systems, techniques and articles are also described.

RELATED APPLICATION

This application claims priority to U.S. Pat. App. Ser. No. 61/402,076filed on Aug. 23, 2010, the contents of which are hereby fullyincorporated by reference.

TECHNICAL FIELD

The subject matter described herein relates to systems and techniquesfor the collection and processing of oleaginous microorganisms forapplication such as biooil that uses ultrasound and acoustophoresis.

BACKGROUND

Biofuels, such as biodiesel, that can be produced from biooil feedstocksthat are in turn produced by oleaginous microorganisms, such asmicroalgae, bacillus, fungi, and yeast are increasingly being adopted.Oleaginous microorganisms are microbial with lipid content typically inexcess of 20%. A renewable liquid fuel energy source could play asignificant role in reducing our national dependence on foreign oilimports. Reported in the literature is that oleaginous yeasts andmicroalgae can grow and accumulate significant amounts of lipids (see A.Banerjee, R. Sharma, Y. Chisti and U. C. Banerjee, “BotryococcusBraunii: A renewable source of hydrocarbons and other chemicals”Critical Reviews in Biotechnology, 22 (3), 245-279, 2002; Y. Chisti,“Biodiesel from microalgae beats bioethanol” Trends in Biotechnology, 26(3), 126-131, 2007; P. Metzger and C. Largeau, “Botryococcus braunii: arich source for hydrocarbons and related ether lipids” Appl. Microbiol.Biotechnol, 66, 486-496, 2005; X. Meng, J. Yang, X. Xu, L. Zhang, Q.Nie, M. Xian “Biodiesel production from oleaginous microorganisms”Renewable Energy, 34, 1-5, 2009, the contents of each of theaforementioned papers being incorporated by reference). The oil contentand composition are a function of the type of microorganisms used andthe conditions in which the culturing took place. As an example,microalgae are sunlight driven cell factories that convert carbondioxide to potential biofuels. Microalgae grow at a very fast pace,doubling their biomass within a 24 hour time period and are rich in oil.The lipid content of microalgae can be as high as 70%. In particular,microalgae are reported to be excellent candidates for biodieselproduction because of their higher biomass production, higherphotosynthetic efficiency, and faster growth compared to most otherenergy crops.

Most of the work reported in the literature on the development ofmicrobial oil production has focused on the identification of betterstrains of oleaginous microorganisms, on genetic and metabolicengineering of strains, on the development of the optimal environmentalconditions for microorganism growth, and on the development of theoptimal energy sources to fuel the growth of the microorganisms.

Similar to the biofuel studies there have been studies on chemical andnutraceutical production in microalgae (see J. N. Rosenberg, G. A.Oyler, L. Wilkinson and M. J. Betenbaugh, “A green light for engineeredalgae: redirection metabolism to fuel a biotechnology revolution”,Current Opinion in Biotechnology, 19, 430-436, 2008, the contents ofwhich are hereby incorporated by reference). However there has beenlittle effort spent on the harvesting of microorganisms, particularlyfrom large-scale (100 liter to 2 million liter) volume cultures.Therefore, significant challenges remain in the energy efficient andeconomical harvesting of microorganisms from their host medium, as wellas steps to collect the microbial oils. In particular, harvesting of themicroorganisms by the concentration and separation of the microorganismsfrom their host medium, typically water.

Algae use in bioreactors or large ponds is increasingly being employedfor biofuels and nutraceuticals. Metzger, and Largeau (2005) andBanerjee (2002) describe the use of Botryococcus braunii as a source ofhydrocarbons and similar lipids, such as C₂₇diane, C₃₀ botryococcene,squalene, tetramethylsqualene and trs,trs-lycopadine, among othersincluding ether lipids, epoxides and sterols. Weldy and Huesemann (C. S.Weldy and M. Huesemann, “Lipid production by Dunaliella salina in batchculture: effects of nitrogen limitation and light intensity” U.S.Department of Energy Journal of Undergraduate Research, Vol. VII,115-122, 2007) and Hejazi and Wijffels (M. A. Hejazi and R. H. Wijffels,“Effect of light intensity on beta-carotene production and extraction byDunaliella salina in two-phase bioreactors” Biomolecular Engineering,20, 171-175, 2003) describe the use of Dunaliella salina, for lipidproduction and beta-carotene production. Other researchers, includingChisti (2007), Meng et al. (2009) and flu (Q. Hu, M. Sommerfeld, E.Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, and A. Darzins,“Microalgae triacylglycerols as feedstocks for biofuel production:perspectives and advances” The Plant Journal, 54, 621-639, 2008) discussthe use of microalgae for biodiesel and triacylglycerols production.Lastly, Rosenberg et al (2008) describe a whole list of nutraceuticals,pharmaceuticals, and high-value chemicals produced from microalgae. Inall these applications, there is a need for improved algaeconcentrating, or as is known, dewatering. Conventional techniquesinvolve batch centrifuging at high-cost.

SUMMARY

In one aspect, an apparatus is provided that includes a microorganismcollection and separation unit, a lipid extraction unit, and a lipidcollection and separation unit. The microorganism collection andseparation unit can include a first flow chamber that in turn comprisesa first inlet through which is flowed a mixture of a host fluid andmicroorganisms along a first flow path, a first outlet, and at least onefirst ultrasonic transducer forming a standing acoustic wavesubstantially perpendicular to the first flow path to selectivelyseparate the microrganisms from the host fluid so that suchmicroorganisms are collected and remaining host fluid exits the firstflow chamber via the first outlet. The lipid extraction unit can includea second flow chamber that in turn comprises a second inlet throughwhich is flowed a mixture of a host fluid and microorganisms collectedby the microorganism and separation unit along a second flow path, asecond outlet, and at least one second ultrasonic transducer forming astanding acoustic wave substantially perpendicular to the second flowpath to selectively rupture cellular walls and membranes of themicrorganisms to release lipids, the lipids being collected andremaining host fluid exiting the second flow chamber via the secondoutlet. The lipid collection and separation unit can include a thirdflow chamber that in turn comprises a third inlet through which isflowed a mixture of a host fluid and lipids from the lipid extractionunit along a third flow path, a third outlet, and at least one thirdultrasonic transducer forming a standing acoustic wave substantiallyperpendicular to the third flow path to selectively separate the lipidsfrom the host fluid so that such lipids are collected and remaining hostfluid exits the third flow chamber via the third outlet.

The standing acoustic wave can direct the microorganisms to at least onecollection pocket for collection and removal from the first flowchamber. Similarly, the standing acoustic wave directs the lipids to atleast one collection pocket for collection and removal from the thirdflow chamber.

The microorganisms can be selected from a group consisting of:microalgae, yeast, fungi, bacteria, and spores. Each transducer can beoptimized for a specific range of particles selected from a groupconsisting of microalgae, yeast, fungi, bacteria, and spores.

One or more of the first, second, and/or third ultrasonic transducer(s)can operate at a frequency in a range of 1 MHz to 10 MHz. The ultrasonictransducers can be driven at a constant frequency of excitation or afrequency sweep pattern. The second transducer can be driven by a pulsedwaveform that does not result in cavitation of the microorganisms.Conversly, the second ultrasonic transducer can be driven by a waveform(e.g., an arbitrary waveform) that results in cavitation of themicroorganisms. One or more of the first, second, and/or thirdultrasonic transducer(s) can be embedded in a wall of the correspondingflow chamber.

The lipid extraction unit can also include a recirculation unitcomprising a tank, an inlet, and outlet, and at least one recirculationarm. The tank can include at least one plate transducer and/or at leastone array transducer. The recirculation arm can include a flattransducer and/or a ring transducer. The lipid collection and separationunit can cause the lipids to agglomerate such that their buoyancy forceis sufficient to force the lipids to float to the top of the third flowchamber to result in a lipid layer which can then be collected.

In an interrelated aspect, a method is provided in which microorganismsare collected and separated from a host medium (e.g., water, etc.) usinga microorganism collection and separation unit. Thereafter, cellularwalls and membranes of the microorganisms are ruptured using a lipidextraction unit in order to release their lipids. Subsequently, thelipids are collected and separated from the host medium using a lipidcollection and separation unit.

In a further interrelated aspect, a system is provided that comprisesthree subsystems including a first subsystem for the trapping,concentration, collection, and separation of microorganisms such asmicroalgae, yeast, fungi, bacteria, or spores from a host medium such aswater, a second subsystem for the rupturing of the cell wall andcellular membranes of the microorganism such that the lipid content ofthe microorganism is released into the water, typically in the form ofmicroscopic oil droplets, and a third subsystem for the concentration,collection, and separation of oil droplets from an oil/water emulsion,where the oil droplets are the lipids of said microorganisms, and wherethe separation of the oil droplets results in an oil layer, which canthen be harvested as a feedstock for the production of biofuels or forother uses (e.g., carotenes as food supplements). The first subsystem totrap, concentrate, collect, and separate the microorganisms from wateremploys a flow chamber through which the mixture flows, and anultrasonic transducer, embedded in the flow chamber wall, which, incombination with an acoustic reflector typically located at the wallopposite the transducer face, generates an acoustic standing wave in thewater and acts as a trap for the microorganisms in the acoustic fieldthrough the action of the acoustic radiation force. The second subsystememploys an ultrasonic transducer, embedded in the wall of a tank or aflow through channel, which generates high intensity ultrasound with orwithout cavitation effects sufficient to rupture the cell wall of themicroorganisms resulting in the release of the lipid content of themicroorganism into the water, resulting in an oil/water emulsion. Thethird subsystem employs a flow-through chamber through which the oilwater emulsion flows and an ultrasonic transducer embedded in the flowchamber wall, which, in combination with an acoustic reflector typicallylocated at the wall opposite the transducer face, generates an acousticstanding wave in the emulsion that acts as a trap for the microscopicoil droplets through the action of the acoustic radiation force. Thetrapping of the oil droplets results in the coalescence and aggregationof the oil droplets into large aggregates of oil droplets such thatbuoyancy forces the aggregates to rise to the top of the flow channel,where the lipids form an oil layer which can then be harvested asfeedstock for the production of biofuels or for other uses (e.g.,carotenes as food supplements).

The current subject matter provides many advantages. For example, thecurrent subject matter enables the processing of large quantities of ahost medium, e.g., water, which is laden with oleaginous microorganismsby efficiently trapping, concentrating, and separating themicroorganisms from the host medium. This is accomplished by rupturingthe cell walls and cellular membranes of the microorganisms so that themicrobial lipids (i.e., biooils, etc.) that are contained within theoleaginous microorganisms are released into the host medium.

The details of one or more variations of the subject matter describedherein are set forth in the accompanying drawings and the descriptionbelow. Other features and advantages of the subject matter describedherein will be apparent from the description and drawings, and from theclaims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating calculated acoustic force operating onmicron-size particles as a function of the particle (or droplet) radiusat a frequency of 1 MHz and acoustic pressure amplitude of 0.5 MPa.

FIG. 2 is a photomicrograph of acoustophoretic trapping of the algaeDunaliella salina in flowing water in which the transducer is at thetop, just out of the image; the column of trapped algae is about 2.5 cmhigh×1 cm wide, and where the ultrasonic pressure nodes are seen as thehorizontal planes in which the algal cells are captured; the water flowis from left to right.

FIG. 3 is a block diagram illustrating a system including threesub-systems, namely a microorganism concentration and separation unit310, a lipid extraction unit 320, and a lipid collection and separationunit 330.

FIG. 4 is a diagram illustrating an apparatus having flow channels,acoustic transducer, reflective surface, and collection pocket, for theharvesting of microalgae through acoustophoretic trapping; thetransducer is a 2 MHz PZT-4 transducers; the direction of the fluid flowis horizontal and the direction of the acoustic field is vertical.

FIG. 5 is a photograph (at 10× magnification) of a typical collection ofmicroalgae obtained using an apparatus such as illustrated in FIG. 4;the fluid flow direction is horizontal and the acoustic standing wave isin the vertical direction.

FIG. 6 is a series of three photos (at 10× magnification) ofgravitational settling of the microalgae after the fluid flow has beenstopped and the acoustic field has been turned off with the arrowsindicate progression of time over 1 second intervals.

FIG. 7 is a photograph (at 10× magnification) showing cavitationoccurring; the process is used to rupture the cell walls and thecellular membranes of the microalgae; cavitation is evidenced by thebubbles that form in the dispersion and have risen to the surface.

FIG. 8 is a photograph (at 400× magnification) of an oil/water emulsionobtained as a result of the cavitation process applied to a suspensionof microalgae; typical oil droplet diameter is on the order of 3 μm.

FIG. 9 is a photograph (at 400× magnification) of a stable emulsion madefrom 400 ml water, 10 ml baby oil, and four tablets of Ceteareth-20.

FIG. 10 is a photograph of an apparatus for oil concentration andseparation; the stable emulsion flows through the region of the acousticfield in a downward vertical direction; the acoustic field is in thehorizontal direction.

FIG. 11 is a series of four photos (at 10× magnification) showing theformation of oil droplet aggregates as a result of the trapping of theoil droplets in the acoustic field; the top-most photograph is the firstin the time series; the bigger chain of oil droplets, formed as resultof coalescence and agglomeration, has just started to rises as a resultof buoyancy, and can be seen completely separated from the smaller lineof oil droplets in the final, bottom-most photograph.

FIG. 12 is a photograph (10× magnification) of the collected oil layerat the top of the flow chamber as a result of the coalescence,aggregation, and concentration of the oil droplets.

FIG. 13 is a diagram illustrating an apparatus for trapping,concentration, and collection of microorganisms and their separationfrom the host medium.

FIG. 14 is a diagram illustrating a frequency sweep pattern that can beused to translate trapped particles along the direction of the acousticfield.

FIG. 15 is a diagram illustrating an apparatus for trapping,concentration, and collection of microorganisms and their separationfrom the host medium, containing multiple transducers in line.

FIG. 16 is a diagram illustrating an apparatus for trapping,concentration, and separation of lipids/biooils from an oil/wateremulsion.

FIG. 17 is a diagram illustrating a pulsed waveform that can be used inthe rupturing process of the cellular walls and membranes of themicroorganisms.

FIG. 18 is a diagram illustrating an arbitrary waveform that can be usedin the rupturing process of the cell wall of the microorganisms.

FIGS. 19A-D are diagrams illustrating variations of an apparatus for theprocessing of microorganisms.

DETAILED DESCRIPTION

The current subject matter utilizes acoustophoresis, a low-power,no-pressure-drop, no-clog solid-state approach to particle removal fromfluid dispersions: i.e., it is used to achieve separations that are moretypically performed with porous filters and centrifuges, but it has noneof the disadvantages of these systems. For example, the diagram 100 ofFIG. 1 shows the forces for an applied acoustic frequency of 1 MHz(typical for an ultrasonic transducer) and an acoustic pressure of 0.5MPa maximum at the antinodes (readily achieved in water). Achievement ofhigher applied acoustic frequencies and higher acoustic pressures willrequire better impedance matching. Examples of acoustic filtersutilizing acoustophoresis can be found in commonly owned U.S. patentapplication Ser. Nos. 12/947,757, 61/261,686, 13/085,299 and 61/342,307,the contents of all of these applications are hereby fully incorporatedby reference.

The acoustic radiation force (F_(ac)) acts on the secondary-phaseparticles (or fluid droplets), pushing them to the nodes (or antinodes)of the acoustic standing wave. The magnitude of the force depends on theparticle density and compressibility relative to the fluid medium, andincreases with the particle volume. The diagram 100 of FIG. 1illustrates the acoustic force that operates on four different secondaryphases in water as a function of the particle (or droplet) radius. Thefour secondary phases are hexanes (a mixture of hydrocarbons, a modelfor oils), red blood cells (a model for biological cells), bacterialspores (a model for “large” protein clusters and polystyrene beads suchas are used for flow cytometry), and paramagnetic polystyrene beads(used for various biological capture and separation protocols).Parameters used in the calculation of the acoustic force are given beloware in Table 1 (which are of particular interest regarding the algaeparameters).

The current subject matter is advantageous in that it usesacoustophoresis for separations in extremely high volumes and in flowingsystems with very high flow rates. Separations have been done formicron-size particles, for which the acoustophoretic force is quitesmall. For example, B. Lipkens, J. Dionne, A. Trask, B. Szczur, A.Stevens, E. Rietman , “Separation of micron-sized particles inmacro-scale cavities by ultrasonic standing waves,” Presented at theInternational Congress on Ultrasonics, Santiago, Jan. 11-17, 2009; andB. Lipkens, J. Dionne, M. Costolo, A. Stevens, and E. Rietman,“Separation of bacterial spores from flowing water in macro-scalecavities by ultrasonic standing waves”, (Arxiv) June 2010, the contentsof both papers are hereby fully incorporated by reference) show thatBacillus cereus bacterial spores (a model for anthrax) have been trappedat 15% efficiency in an acoustophoretic cavity embedded in a flow systemthat can process drinking water at rates up to 120 mL/minute (1cm/second linear flow). The concentration ratio has been as high as 1000in a single-pass, small-scale prototype acoustocollector. However, thetechniques described in this paper do not always scale up to higher flowrates.

An acoustophoretic separator can be created by using a piezoelectricacoustic transducer and an opposing reflection surface (or a secondtransducer) to set up a resonant standing wave in the fluid of interest.The ultrasonic standing waves create localized regions of high and lowpressure, corresponding to high and low density of the fluid. Secondaryphase contaminants are pushed to the standing wave nodes or antinodesdepending on their compressibility and density relative to thesurrounding fluid. Particles of higher density and compressibility(e.g., bacterial spores) move to the nodes in the standing waves;secondary phases of lower density (such as oils) move to the antinodes.The force exerted on the particles also depends on their size, withlarger particles experiencing larger forces.

Diagram 200 of FIG. 2 shows the acoustophoretic collection of algae in aflowing water stream. A flat, circular transducer can, for example, beused in an acoustocollector to generate the collected matter in FIG. 1.The pressure field of such a transducer is a Bessel function that has aradial component in addition to the linear standing wave. The radialcomponent acts to hold the captured algae in the column against thefluid flow. The trapped algae are then further concentrated in region bygravitational settling or by being driven to a collector pocket througha slow frequency sweeping method similar to that given in (i) B.Lipkens, M. Costolo, and E. Rietman , “The effect of frequency sweepingand fluid flow on particle trajectories in ultrasonic standing waves”,IEEE Sensors Journal, Vol. 8, No. 6, pp. 667-677, 2008; (ii) Lipkens, J.Dionne, M. Costolo, and E. Rietman, “Frequency sweeping and fluid floweffects on particle trajectories in ultrasonic standing waves,”Acoustics 08, Paris, June 29-Jul. 4, 2008; and (iii) B. Lipkens, J.Dionne, A. Trask, B. Szczur, and E. Rietman, “Prediction and measurementof particle velocities in ultrasonic standing waves,” J. Acoust. Soc.Am. 124, No. 4, pp. 2492 (A). The contents of each of the aforementionedpapers are hereby fully incorporated by reference.

Physics of acoustophoresis. Acoustophoresis is the separation of asecond phase (or phases) from a host fluid using sound pressure tocreate the driving force. An ultrasonic transducer operating at a fixedfrequency f (Hz) is used to set up an acoustic standing wave in afluid-filled cavity. The standing wave is characterized by a localpressure p that is a function of position (x) and time (t),

p(x,t)=P cos(kx)sin(ωt)   (1)

where P is the amplitude of the acoustic pressure; k is the wavenumber(=2π/λ, where λ is the wavelength), and ω=2πf, where ω is the angularfrequency. The pressure of the acoustic wave produces an acousticradiation force F_(ac) on secondary-phase elements according to

$\begin{matrix}{{F_{a\; c} = {X\; \pi \; R_{p}^{3}k\; \frac{P^{2}}{\rho_{f}c_{f}^{2}}{\sin \left( {2{kx}} \right)}}},} & (2)\end{matrix}$

where R_(p) is the particle radius, ρ_(f) is the density of the fluidmedium, c_(f) is the speed of sound in the fluid, and Xis the acousticcontrast factor, defined by

$\begin{matrix}{{X = {\frac{1}{3}\left\lbrack {\frac{{5\Lambda} - 2}{1 + {2\Lambda}} - \frac{1}{\sigma^{2}\Lambda}} \right\rbrack}},} & (3)\end{matrix}$

where A is the ratio of the particle density to fluid density and σ isthe ratio of the speed of sound in the particle to the sound speed inthe fluid. The acoustic radiation force acts in the direction of theacoustic field. The acoustic radiation force is proportional to theproduct of acoustic pressure and acoustic pressure gradient. Aninspection of the acoustic radiation force shows that it is proportionalto the particle volume, frequency (or wavenumber), the acoustic energydensity (or the square of the acoustic pressure amplitude), and theacoustic contrast factor. Note also that the spatial dependency hastwice the periodicity of the acoustic field. The acoustic radiationforce is thus a function of two mechanical properties, namely densityand compressibility.

TABLE 1 Properties of water and 4 selected secondary phases. ρ c Λ X(density) (speed of sound) (dimen- (dimen- Material (kg/m³) (m/s)sionless) sionless) Water 1000 1509 — — Hexanes 720 1303 0.72 −0.402Blood Cells 1125 1900 1.125 0.185 Bacterial Spores 1100 1900 1.1 0.173Magnetic beads 2000 1971 2.0 0.436

For three dimensional acoustic fields, a more general approach forcalculating the acoustic radiation force is needed. Gor'kov's (1962)formulation can be used for this (see L. P. Gor'kov, “On the forcesacting on a small particle in an acoustical field in an ideal fluid,”Sov. Phys. Dokl., vol. 6, pp. 773-775, 1962). Gor'kov developed anexpression for the acoustic radiation force F_(ac) applicable to anysound field. The primary acoustic radiation force is defined as afunction of a field potential U, given by

F _(ac)=−∇(U), (4)

where the field potential U is defined as

$\begin{matrix}{{U = {V_{0}\left\lbrack {{\frac{\langle{p^{2}\left( {x,y,t} \right)}\rangle}{2\rho_{f}c_{f}^{2}}f_{1}} - {\frac{3\rho_{f}{\langle{v^{2}\left( {x,y,t} \right)}\rangle}}{4}f_{2}}} \right\rbrack}},} & (5)\end{matrix}$

and f₁ and f₂ are the monopole and dipole contributions defined by

$\begin{matrix}{{f_{1} = {1 - \frac{1}{{\Lambda\sigma}^{2}}}},{f_{2} = \frac{2\left( {\Lambda - 1} \right)}{{2\Lambda} + 1}},} & (6)\end{matrix}$

where p(x,y,z,t) is the acoustic pressure and v(x,y,z,t) is the fluidparticle velocity. V_(o) is the volume of the particle.

The diagram 100 of FIG. 1 shows the force required to separate smallparticles of various material properties. Each material has its own Xparameter given in Equation [3]. In diagram 100, material properties(e.g. speed of sound, density) are used for the indicated material. Thegraph for bacteria spore is also valid for other materials of similarbulk modulus. Meaning smaller bacteria spore, very large proteinclusters, and polystyrene microspheres would all be in this category.The blood cell curve is for any cells of similar bulk modulus. Finallythe hexane curve would be valid for any tiny drops of oil-like materialwith the radius indicated on the curve. These curves are for, as anexample, 1 MHz applied acoustic frequency and an acoustic pressure of0.5 MPa. These are easily achieved control variables. Higher frequencyand higher pressure require better impedance matching and will affordbetter separation of smaller particles—down to 10 s of nm.

FIG. 3 illustrates an overall system 300 to collect and processoleaginous microalgae for the production of biofuels that comprisesthree sub-systems. A microorganism concentration and separation unit 310acoustophoretically concentrates and separates microorganisms from ahost medium such as water. A lipid extraction unit 320 applies highintensity ultrasound to rupture the cell walls and cellular membranes ofthe microorganisms so that the lipid (i.e., biooil, etc.) content of themicroorganisms is released into the water and an oil/water emulsion isformed. A lipid collection and separation unit 330 acoustophoreticallyconcentrates and separates the water from the lipids, which werereleased by the microorganisms into water. The resulting oil layer canthen be harvested for use as a feedstock for the production of biofuelsor for other uses (e.g., carotenes as food supplements). While thecurrent subject matter is mainly directed to microalgae, it isapplicable to other types of microorganisms.

With regard to the microorganism concentration and separation unit 310,algae of the halophilic Dunaliella Salina were grown in a bottle filledwith salt water and placed under a grow light. The algae were removedfrom the bottle through tubes that passed them into a flow channel andpast an acoustic transducer. A sample apparatus is illustrated indiagram 400 of FIG. 4. With this arrangement, the flow chamber ishorizontal with the transducer on top facing downward. Therefore, theresulting acoustic standing wave was in the vertical direction. Thetransducer was a PZT-4 2 MHZ transducer. A peristaltic pump was used togenerate fluid flow rates that are most typically about 50 ml/min.

The acoustic transducer was connected to an amplifier which received itssignal from a function generator and operated at about 15 Vrms. Once thefluid flow and the acoustic transducer were turned on, trapping andconcentration of microalgae took place instantaneously. The microalgaewere trapped in the acoustic field against the fluid drag force by meansof the action of the acoustic radiation force. The collection ofmicroalgae continued over time and eventually, typically after severalminutes, large, beam-like collections of microalgae were seen in theregion between the transducer face and the opposition reflective wall. Atypical result of the acoustic trapping of microalgae for about 15 to 20minutes in the system of FIG. 4 is shown in diagram 500 of FIG. 5.

Two methods for the further separation and collection of the microalgaehave been used, one is gravitational settling once the fluid flow hasbeen stopped and the acoustic field has been turned off, as shown indiagram 600 of FIG. 6, and the second is the use of a frequency sweepmethod (see, for example, B. Lipkens, M. Costolo, and E. Rietman , “Theeffect of frequency sweeping and fluid flow on particle trajectories inultrasonic standing waves”, IEEE Sensors Journal, Vol. 8, No. 6, pp.667-677, 2008) to translate and collect the microalgae in a collectorpocket. For the first method the acoustic field has to be oriented alongthe vertical direction, for the second method there is no orientationconstraint.

In one implementation of the microorganism concentration and separationunit 310, a flow channel within a flow chamber can be used to flow thefluid dispersion, typically water and a secondary-phase component thatis dispersed in the water. See, for example, the diagram 1300 of FIG. 13which illustrates a flow chamber 1302 having an inlet 1301 and an outlet1304, at least one transducer 1303, and at least one correspondingreflector 1305. The secondary-phase component in this case is themicroorganism of interest, e.g., microalgae. At least one ultrasonictransducer can be located in the wall of the flow channel. Piezoelectrictransducers are often used. The transducer can be driven by anoscillating voltage that has an oscillation at an ultrasonic frequency.The ultrasonic frequency is typically in the range of several Megahertzand the voltage amplitude is on the order of tens of volts. Thetransducer, in combination with an acoustic reflection surface locatedat the wall of the flow tube opposite to the transducer, serves togenerate an acoustic standing wave across the flow channel. Typicalpressure amplitudes in the region of the acoustic standing wave or fieldare on the order of 0.5 MPa, amplitudes readily available withconventional piezoelectric transducers. The pressure amplitudes arebelow the cavitation threshold values so that a high intensity standingwave field is created without generation of cavitation effect orsignificant acoustic streaming. Acoustic streaming refers to atime-averaged flow of the water produced by the sound field. Typically,when acoustic streaming is generated it results in circulatory motionthat may cause stirring in the water. Cavitation typically occurs whenthere are gas bodies, such as air micro-bubbles, present in the water.The effect of the sound pressure is to create micro-bubble oscillationswhich lead to micro-streaming and radiation forces. Micro-streamingaround bubbles lead to shearing flow in the surround liquid. This flowcontains significant velocity gradients. If a microorganism is locatedin this shearing flow, the uneven distribution of forces on the cellwalls can lead to significant shear stresses exerted on the cell wallsthat may lead to cell wall disruption and rupture. At higher soundintensity levels, the micro-bubble oscillations become more intense, andthe bubble can collapse leading to shock wave generation and freeradical production. This is termed inertial cavitation.

The acoustophoretic force created by the acoustic standing wave on thesecondary phase component, i.e., the microorganism, is sufficient toovercome the fluid drag force. In other words, the acoustophoretic forceacts as mechanism that traps the microorganisms in the acoustic field.The acoustophoretic force drives the microorganisms to the stablelocations of minimum acoustophoretic force amplitudes. Over time thecollection of microorganisms grows steadily. Within minutes, dependingon the concentration of the secondary phase component, the collection ofmicroorganisms takes on the shapes of a beam-like collection ofmicroorganisms consisting of disk-shaped collections of microorganisms,each disk spaced by a half wavelength of the acoustic field. The beam ofdisk-shaped collections of microorganisms is stacked between thetransducer and the opposing, acoustically-reflective flow-tube wall.Therefore, acoustophoretic forces are able to trap and concentratemicroorganisms in the region of the acoustic field while the host mediumcontinues to flow past the concentrated microorganisms. The collectionof microorganisms can continue until very large volumes of the hostmedium have been flowed through the trapping region and the capture ofthe containing microalgae has been attained. Further separation of theconcentrated microorganisms from the host medium is achieved by twomeans. For a horizontal flow of the host medium, gravitational settlingmay be used to drive the concentrated microorganisms into collectorpockets (see, for example, a collection pocket as illustrated in diagram1500 of FIG. 15). For vertical or horizontal flow of the host medium, aslow frequency sweeping method may be used to translate themicroorganisms into collector pockets (see, for example, diagram 1400 ofFIG. 14). In this method, the frequency of the acoustic standing wave isslowly swept over a small frequency range, which spans at least a rangeof two frequencies corresponding to the one lower than the and onehigher than the resonance of the standing wave mode of the cavity. Thesweep period is typically on the order of seconds. This frequencysweeping method will slowly translate the collected microorganisms inthe direction of the acoustic field towards one of the walls of the flowchamber where the microorganism may be collected for further processing.It will be appreciated that an array or differing types of transducerscan be used (which in turn may operate at different or varying resonancefrequencies).

With regard to the lipid extraction unit 320, two approaches can be usedto extract the oil content from the microalgae. The first method isultrasonic cavitation. The second method is the use of ultrasound ofhigh intensity but not of cavitating amplitude to break the cell walland cellular membranes of the microalgae (using, for example, anarbitrary waveform such as that illustrated in diagram 1800 of FIG. 18).A proof-of-concept demonstration was conducted in which a suspension ofconcentrated microalgae was put into a glass tube, six inches long andoriented vertically. A PZT-4 2.3 MHz transducer was mounted to thebottom. This system was used to cavitate the suspension of themicroalgae in water, as shown in diagram 700 of FIG. 7. During thecavitation process, the cell wall and cellular membranes were rupturesand broken and the lipids were released from the cells. Typically, theacoustic field that results in cavitation was applied for about fiveminutes. Within a few minutes most of the microalgae debris—the cellwall and cellular debris which is darker green and light brown incolor—falls to the bottom of the tube. The remaining dispersion was aclear, light green mixture. The lipids (oil) and the water are now in anemulsion, as seen in diagram 800 of FIG. 8. Typical oil droplet size wason the order of 3 μm in diameter.

The lipid extraction unit 320 comprises a vessel that is configured torupture of the cell walls and cellular membranes of the microorganismsto release their lipid content. See, for example, diagram 1600 of FIG.16 which provides a system including a flow chamber 1603 having at leastone inlet 1601, a water outlet 1602, and a primary outlet 1605, at leastone transducer 1604, and at least one corresponding reflector (notshown) that is on the wall opposing the transducer. In the wall of thevessel holding the microorganisms is at least ultrasonic transducer. Thetransducer can be driven by an oscillating voltage signal at ultrasonicfrequencies typically in the kilohertz to Megahertz range. In oneimplementation, the transducer can be driven at voltages that generateacoustic standing waves of sufficient amplitude such that cavitation isgenerated. The result of cavitation occurring on the cell walls andmembranes of the microorganisms is the generation of large shear forcesof sufficient amplitude to rupture the cell wall and cellular membranesof the microorganisms. Once the cell wall is ruptured, the lipidcontent, i.e, the biooil, is released into the host medium, i.e., thewater. This process results in an oil/water emulsion that also containsthe cellular debris.

In another implementation of the lipid extraction unit 320, thetransducer can be driven by a pulsed voltage signal consisting ofshort-duration, large, positive-amplitude voltage spikes, followed by alonger duration of no applied voltage signal (see, for example, diagram1700 of FIG. 17). This pulsed pattern can then repeated according to apre-defined repetition rate or period. The effect of this excitation isto generate very large amplitude compressive pressure pulses in waterthat are sufficient to rupture the cell walls and cellular membranes ofthe microorganisms.

In another variation of the lipid extraction unit 320, the transducercan be driven by a pulsed voltage signal consisting of short-duration,large, negative-amplitude voltage spikes, followed by a longer durationof no applied voltage signal. This pulsed pattern can then be repeatedaccording to a pre-defined repetition rate or period. The effect of thisexcitation is to generate very large amplitude expansion-type pressurepulses in water that are sufficient to rupture the cell walls andcellular membranes of the microorganisms.

The lipid extraction unit 320 can optionally include one or more varietyof tanks such as those as shown in diagrams 1900-1930 of FIGS. 19A-19D.In the top two arrangements 1900, 1910, a recirculation system 1903 isemployed in which a transducer 1901 (a flat transducer) or 1902 (a ringtransducer) is within a tubular member extending from and back into atank 1906. Within the tank 1906, host fluid enters via an inlet 1905 andexits via an outlet 1907 (such arrangement can be reversed depending onthe desired configuration). In addition, within the tank 1906 there canbe a plate transducer 1909 and/or an array transducer 1908 to furtherexpose the host fluid to high intensity ultrasound.

With regard to the lipid collection and separation unit 330, a thirdproof-of-concept demonstration was conducted that demonstrated thecoalescence, aggregation, concentration and separation of oil dropletsfrom a stable oil/water emulsion. An emulsion was created to simulate anemulsion of microalgae lipids in water. A stable emulsion was createdusing water, baby oil, and Ceteareth-20. A fluid-flow apparatus was thenused to separate the components of the emulsion, resulting in an oillayer and a water layer that are separate from one another.

A stable emulsion was created from a mixture of four tablets ofCeteareth-20 (a common emulsifier), 400 mL of hot (180° F.) water, and10 ml of baby oil. A photo, taken at 400× magnification, of the stableemulsion is shown in diagram 900 of FIG. 9. The oil droplets in thestable emulsion ranged in diameters from about three to six μm.

Next, a flow-through apparatus was used to concentrate and separate theoil phase from the emulsion. A photograph of the apparatus is shown indiagram 1000 of FIG. 10. The emulsion is flowing in a downward verticaldirection. The acoustic field is perpendicular to the flow field, andacoustophoresis is used to trap the oil particles.

The transducer was a 2 MHz PZT-4 transducers, operating at 2 MHz and anapplied voltage about 15 Vrms. The flow rate of the emulsion through theflow apparatus was on the order of 200 ml/min. After a typical trappingtime of five minutes the fluid flow was stopped and the height of theoil layer that had been collected at the top of the chamber wasmeasured.

Diagram 1100 of FIG. 11 shows the formation of oil droplets trapped inthe acoustic field. Once the oil droplets are trapped, they coalesce toform bigger droplets, and agglomerate to form aggregates of thedroplets. Once the aggregates have grown to a sufficient size, theirbuoyancy force drives the oil droplet aggregates to the surface of thechamber. Continuous formation of oil droplet aggregates is observed,followed by the rapid translation of the aggregates as a result ofbuoyancy. A second observation indicating rapid separation of the oildroplets from the water is from the visual observation of a cloudysolution above the transducer, (i.e., where the unprocessed emulsion hasnot yet passed through the acoustic field, but of a very clear solutionbelow the transducer, where the oil has been removed by the acousticfield). These regions above and below the acoustic trapping region areseparated by a sharp line between the cloudy solution and clearsolution. After about 5 minutes of application of an acoustic trappingfield while flowing the emulsion through the system, a layer ofcollected oil droplets is observed at the top of the chamber, as shownin diagram 1200 of FIG. 12.

The lipid collection and separation unit 330 can also include a flowchannel is used to flow the oil/water emulsion. The flow direction ofthe emulsion is typically in the downward vertical direction. At leastone ultrasonic transducer (e.g., a piezoelectric transducer, etc.) canbe located in the wall of the flow channel and e driven by anoscillating voltage operating at an ultrasonic frequency, typically inthe range of several Megahertz, and with voltage amplitude on the orderof tens of volts. The transducer, in combination with an acousticreflector located at the opposing wall of the flow tube, generates anacoustic standing wave across the flow channel. Typical pressureamplitudes are on the order of 0.5 MPa, amplitudes that are readilyavailable with conventional piezoelectric transducers. The pressureamplitudes are below the cavitation threshold values so that ahigh-intensity standing-wave acoustic field is created withoutgeneration of cavitation effect or significant acoustic streaming. Theacoustophoretic force created by the acoustic standing wave on thesecondary phase component, i.e., the oil droplets, is sufficient toovercome the fluid drag force. In other words, the acoustophoretic forceacts as mechanism that traps the oil droplets in the acoustic field. Theacoustophoretic force drives the oil droplets to the stable locations ofminimum acoustophoretic force amplitudes. Within seconds, depending onthe concentration, the oil droplets form beam-like striations consistingof disk-shaped aggregates of oil droplets, each disk spaced by a halfwavelength of the acoustic field; the disks are stacked between thetransducer and the acoustic reflector. As soon as the oil aggregatesreach a critical volume, the buoyancy force that the aggregateexperiences is sufficient to drive the aggregates to the top of thefluid layer. Therefore, the acoustophoretic force acts as a concentratorof the oil droplets, causing coalescence and agglomeration of thedroplets, and turning them into large aggregates of oil droplets, atwhich points buoyancy forces the oil aggregates to rise. Over time, asteadily increasing layer of separated oil, i.e., lipids, is collectedat the top of the flow chamber. Various techniques can be employed toremove the oil layer.

While this specification contains many specifics, these should not beconstrued as limitations on the scope of what is claimed or of what maybe claimed, but rather as descriptions of features specific toparticular variations. Certain features that are described in thisspecification in the context of separate variations can also beimplemented in combination in a single variation. Conversely, variousfeatures that are described in the context of a single variation canalso be implemented in multiple variations separately or in any suitablesub-combination. Moreover, although features may be described above asacting in certain combinations and even initially claimed as such, oneor more features from a claimed combination can in some cases be excisedfrom the combination, and the claimed combination may be directed to asub-combination or a variation of a sub-combination. Similarly, whileoperations are depicted in the drawings in a particular order, thisshould not be understood as requiring that such operations be performedin the particular order shown or in sequential order, or that allillustrated operations be performed, to achieve desirable results. Onlya few examples and implementations are disclosed. Variations,modifications and enhancements to the described examples andimplementations and other implementations may be made based on what isdisclosed.

1. An apparatus comprising: a microorganism collection and separationunit comprising: a first flow chamber comprising: a first inlet throughwhich is flowed a mixture of a host fluid and microorganisms along afirst flow path; a first outlet; and at least one first ultrasonictransducer forming a standing acoustic wave substantially perpendicularto the first flow path to selectively separate the microorganisms fromthe host fluid so that such microorganisms are collected and remaininghost fluid exits the first flow chamber via the first outlet; a lipidextraction unit comprising: a second flow chamber comprising: a secondinlet through which is flowed a mixture of a host fluid andmicroorganisms collected by the microorganism and separation unit alonga second flow path; a second outlet; and at least one second ultrasonictransducer forming a standing acoustic wave substantially perpendicularto the second flow path to selectively rupture cellular walls andmembranes of the microorganisms to release lipids, the lipids beingcollected and remaining host fluid exiting the second flow chamber viathe second outlet; and a lipid collection and separation unitcomprising: a third flow chamber comprising: a third inlet through whichis flowed a mixture of a host fluid and lipids from the lipid extractionunit along a third flow path; a third outlet; and at least one thirdultrasonic transducer forming a standing acoustic wave substantiallyperpendicular to the third flow path to selectively separate the lipidsfrom the host fluid so that such lipids are collected and remaining hostfluid exits the third flow chamber via the third outlet.
 2. An apparatusas in claim 1, wherein the standing acoustic wave directs themicroorganisms to at least one collection pocket for collection andremoval from the first flow chamber.
 3. An apparatus as in claim 1,wherein the standing acoustic wave directs the lipids to at least onecollection pocket for collection and removal from the third flowchamber.
 4. An apparatus as in claim 1, wherein the microorganisms areselected from a group consisting of: microalgae, yeast, fungi, bacteria,and spores.
 5. An apparatus as in claim 1, wherein the at least onefirst ultrasonic transducer, the at least one second ultrasonictransducer, and/or the at least one third ultrasonic transducer operateat a frequency in a range of 1 MHz to 10 MHz.
 6. An apparatus as inclaim 1, wherein the at least one first ultrasonic transducer, the atleast one second ultrasonic transducer, and/or the at least one thirdultrasonic transducer are embedded in a wall of the corresponding flowchamber.
 7. An apparatus as in claim 1, wherein the at least one firstultrasonic transducer, the at least one second ultrasonic transducer,and/or the at least one third ultrasonic transducer are driven at aconstant frequency of excitation.
 8. An apparatus as in claim 1, whereinthe at least one first ultrasonic transducer, the at least one secondultrasonic transducer, and/or the at least one third ultrasonictransducer are driven by a frequency sweep pattern.
 9. An apparatus asin claim 1, wherein the at least one second ultrasonic transducer isdriven by a pulsed waveform that does not result in cavitation of themicroorganisms.
 10. An apparatus as in claim 1, wherein the at least onesecond ultrasonic transducer is driven by a waveform that results incavitation of the microorganisms.
 11. An apparatus as in claim 1,wherein the lipid extraction unit further comprises a recirculation unitcomprising a tank, an inlet, and outlet, and at least one recirculationarm.
 12. An apparatus as in claim 11, wherein the tank comprises atleast one plate transducer.
 13. An apparatus as in claim 11, wherein thetank comprises at least one array transducer.
 14. An apparatus as inclaim 11, wherein the at least one recirculation arm comprises a flattransducer.
 15. An apparatus as in claim 11, wherein the at least onerecirculation arm comprises a ring transducer.
 16. An apparatus as inclaim 1, wherein there are a plurality of transducers and eachtransducer is optimized for a specific range of particles selected froma group consisting of microalgae, yeast, fungi, bacteria, and spores.17. An apparatus as in claim 1, wherein the lipid collection andseparation unit causes the lipids to agglomerate such that theirbuoyancy force is sufficient to force the lipids to float to the top ofthe third flow chamber to result in a lipid layer, the lipid layer beingcollected.
 18. A method comprising: concentrating and separatingmicroorganisms from a host medium using a a microorganism collection andseparation unit; rupturing cellular walls and membranes of themicroorganisms to release their lipids using a lipid extraction unit;and concentrating and separating the lipids from the host medium using alipid collection and separation unit.
 19. A method as in claim 18,wherein: the microorganism collection and separation unit comprises: afirst flow chamber comprising: a first inlet through which is flowed amixture of a host fluid and microorganisms along a first flow path; afirst outlet; and at least one first ultrasonic transducer forming astanding acoustic wave substantially perpendicular to the first flowpath to selectively separate the microorganisms from the host fluid sothat such microorganisms are collected and remaining host fluid exitsthe first flow chamber via the first outlet; the lipid extraction unitcomprises: a second flow chamber comprising: a second inlet throughwhich is flowed a mixture of a host fluid and microorganisms collectedby the microorganism and separation unit along a second flow path; asecond outlet; and at least one second ultrasonic transducer forming astanding acoustic wave substantially perpendicular to the second flowpath to selectively rupture cellular walls and membranes of themicroorganisms to release lipids, the lipids being collected andremaining host fluid exiting the second flow chamber via the secondoutlet; and the lipid collection and separation unit comprises: a thirdflow chamber comprising: a third inlet through which is flowed a mixtureof a host fluid and lipids from the lipid extraction unit along a thirdflow path; a third outlet; and at least one third ultrasonic transducerforming a standing acoustic wave substantially perpendicular to thethird flow path to selectively separate the lipids from the host fluidso that such lipids are collected and remaining host fluid exits thethird flow chamber via the third outlet
 20. A system comprising: meansfor concentrating and separating microorganisms from a host medium;means for rupturing cell walls of the microorganisms to release theirlipids; and means for concentrating and separating the lipids from thehost medium.